Tumor specific human monoclonal antibodies and methods of use

ABSTRACT

The invention provides tumor-specific human monoclonal antibodies and functional fragments. Also provided are nucleic acids encoding tumor-specific human monoclonal antibodies and functional fragments. A method for reducing neoplastic cell proliferation is also provided. The method consists of administering an effective amount of a tumor-specific human monoclonal antibody or functional fragment. Also provided is a method of detecting a neoplastic cell in a sample. The method consists of contacting a cell with a tumor-specific monoclonal antibody or functional fragment and detecting the specific binding of the human monoclonal antibody or functional fragment to the sample.

BACKGROUND OF THE INVENTION

The present invention relates generally to cancer and, morespecifically, to human monoclonal antibodies for the treatment anddiagnosis of cancer.

Cancer is one of the leading causes of death in the United States. Eachyear, more than half a million Americans die from cancer, and more thanone million are newly diagnosed with the disease. In cancer, neoplasticcells escape from their normal growth regulatory mechanisms andproliferate in an uncontrolled fashion, leading to the development of amalignant tumor. Tumor cells can metastasize to secondary sites iftreatment of the primary tumor is either not complete or not initiatedbefore substantial progression of the disease. Early diagnosis andeffective treatment of malignant tumors is therefore essential forsurvival.

The current methods for treating cancer include surgery, radiationtherapy and chemotherapy. A major problem with each of these treatmentsis their lack of specificity for cancer cells. For instance, surgicalremoval of the tumor is often incomplete. Even a few residual neoplasticcells can be lethal, as they can rapidly proliferate and metastasize toother sites. Radiation and chemotherapy also have serious limitations.These therapies target all growing cells of the body, including bothnormal and neoplastic cells. Due to their toxicity to normal tissues,the amount of radiation or chemotherapeutic agent that can be safelyused is often inadequate to kill all neoplastic cells. Additionally,their toxicity to normal tissues is manifested by unpleasant sideeffects, including nausea and hair loss, that severely reduce thequality of life for the cancer patient undergoing these treatments.Clearly, a more selective and effective means of treating cancer isneeded.

Monoclonal antibodies are homogeneous preparations of immunoglobulinproteins that specifically recognize and bind to regions, or epitopes,of their corresponding antigens. Neoplastic cells selectively expressantigens which are not present on normal cells. Thus, monoclonalantibodies can be produced that are directed against tumor-specificantigens. Such tumor-specific antigens can be linked to therapeuticmoieties that kill or arrest the growth of neoplastic cells. Inaddition, monoclonal antibodies can be linked to diagnostic moietiesthat allow the imaging of neoplastic cells. Thus, monoclonal antibodiesdirected against antigens selectively expressed by tumor cells comparedto normal cells can be beneficially used for the early detection andeffective treatment of cancer.

Most current immunotherapeutic strategies for cancer have been oflimited utility due to their reliance on mouse monoclonal antibodies.Mouse monoclonal antibodies can be produced easily and in virtuallyunlimited quantities using hybridoma technology. However, whenadministered to humans, they can be recognized as foreign by the humanimmune system and be neutralized before exerting their therapeuticeffect on the diseased tissue. Furthermore, the murine immune systemoften preferentially recognizes immunodominant epitopes of normal humanantigens present on tumor cells. Thus, human tumor-specific antigensoften fail to generate therapeutically beneficial murine antibodies.

Human monoclonal antibodies can overcome both of these limitations. Mostimportantly, human monoclonal antibodies are not as immunogenic asmurine antibodies. Therefore, tumor-specific human monoclonal antibodieswill be able to more effectively target and eliminate neoplastic cells.Furthermore, the human immune system is less likely to generate antigensagainst epitopes present on normal cells, increasing the odds ofgenerating and successfully identifying tumor-specific antigens.Additionally, the repertoire of the human immune system is differentfrom that of the mouse, containing potentially novel antibodyspecificities.

Current procedures to produce tumor-specific human monoclonal antibodieshave generally started with lymphocytes obtained from tumor-bearingpatients. These procedures rely on the stimulation and expansion oftumor-reactive lymphoctyes in vivo. These procedures are seriouslylimited by the narrow range of antigen specificities of activatedB-cells of cancer patients. As it is clearly not possible to immunizeindividuals in vivo with tumor cells, as one can with mice, it has notbeen possible to readily generate tumor-specific human monoclonalantibodies to any given antigen or tumor cell type. Procedures forgenerating tumor-specific antibodies of any desired specificity would bevery beneficial for effective immunotherapy and immunodiagnosis.

Thus, there exists a need for improved tumor-specific human monoclonalantibodies for the therapy and diagnosis of cancer. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a human monoclonal antibody or functionalfragments thereof, having at least one Complementarity DeterminingRegion (CDR) with substantially the amino acid sequence of a CDR of SEQID NO:2 or SEQ ID NO:4. Also provided is a human monoclonal antibody orfunctional fragment thereof, with at least one CDR having substantiallythe amino acid sequence of a CDR of SEQ ID NO:6 or SEQ ID NO:8. Theinvention also provides human monoclonal antibodies or functionalfragments produced by the cell lines H1140, H2420 or H935. H1140, H2420and H935 hybridoma cell lines are also provided.

The invention further provides a nucleic acid encoding a humanmonoclonal antibody or functional fragment thereof, having a nucleotidesequence encoding substantially the amino acid sequence of at least oneCDR encoded by SEQ ID NO:1 or SEQ ID NO:3. Also provided is a nucleicacid encoding a human monoclonal antibody or functional fragmentthereof, having a nucleotide sequence encoding substantially the aminoacid sequence of at least one CDR encoded by SEQ ID NO:5 or SEQ ID NO:7.

A method for reducing neoplastic cell proliferation is also provided.The method consists of administering an effective amount of a humanmonoclonal antibody or functional fragment of the invention. Alsoprovided is a method of detecting a neoplastic cell in a sample. Themethod consists of contacting a cell with a monoclonal antibody orfunctional fragment of the invention and detecting the specific bindingof the human monoclonal antibody or functional fragment to the sample.The presence or increased level compared to a normal cell of themonoclonal antibody or functional fragment indicates the presence of aneoplastic cell in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of LH11238 and LH13 antibodies to the surfaceof live H3464 cells. FIG. 1A shows fluorescent activated cell sorting(FACS) analysis using LH11238 antibody. FIG. 1B shows FACS analysisusing LH13 antibody.

FIG. 2 shows that LH13 antigen is secreted by H3396 cells. FIG. 2A showsthat conditioned medium from H3396 cells (closed circles) competes forbinding of LH13 antibody to fixed cell monolayers. FIG. 2B shows thatLH13 antigen is secreted by H3396 cells and binds to culture dishes.

FIG. 3 shows the purification of LH13 antigen by anion exchangechromatography on a Q Sepharose column.

FIG. 4 shows that LH13 antigen is susceptible to trypsin andendoglycosidase-F/peptide-N-lycosidase F treatment.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to tumor-specific human monoclonal antibodies.The human monoclonal antibodies of the invention specifically bind toneoplastic cells compared to normal cells, and can be used forselectively targeting tumors. These antibodies are human in origin, andare unlikely to generate an immune response upon administration to ahuman subject. Therefore, these antibodies can be conjugated tocytotoxic or cytostatic agents and used to selectively target cancercells for the elimination of tumors. The tumor-specific human monoclonalantibodies can also be used in diagnostic procedures to identifyneoplastic cells. Early detection of cancer greatly increases thechances of an individual surviving the disease.

In one embodiment, the invention provides methods for generatinghybridomas producing tumor-specific human monoclonal antibodies. Normalhuman splenocytes are immunized in vitro with tumor cells or tumor cellmembranes in a mixed lymphocyte reaction. Such immunized splenocytes arethen immortalized to produce hybridomas providing an unlimited supply oftumor-specific human monoclonal antibodies. Using normal cells as thesource of lymphocytes greatly enlarges the repertoire of differenttumor-specific antibodies that can be obtained for the treatment ofcancer. Additionally, the type of cell or cell membrane used as theantigen in the method of the invention can be varied as needed toefficiently produce antibodies for different human therapeutic anddiagnostic applications.

In another embodiment, the invention is directed to nucleic acidsencoding human tumor-specific monoclonal antibodies. The nucleic acidscan be used to express the encoded human antibodies or fragmentsthereof. Additionally, the encoding nucleic acids can be recombinantlyengineered to produce modified human antibodies which exhibit higheraffinity or higher selectivity for tumor cells or to augment otherfunctional characteristics of the encoded antibodies. Such modifiedantibodies can additionally be constructed to contain othertherapeutically advantageous modifications, such as enhanced associationwith cytotoxic agents or increased stimulation of the immune system.

In a further embodiment, the invention is directed to antigensrecognized by the tumor-specific-human monoclonal antibodies. Theantigens can be used for cancer diagnostic procedures and to developspecific cytotoxic reagents for cancer therapy. Tumor-specific antigenscan also be used as a vaccine and administered to individuals at risk ofcancer to develop an effective immune response against neoplastic cells.The nucleic acids encoding the tumor-specific antigens can be used asprobes in diagnostic procedures, or modified by recombinant methods todevelop specific inhibitors of the antigen.

The basic structure of an immunoglobulin or antibody molecule consistsof two identical light chains and two identical heavy chains, whichassociate non-covalently and can also be linked by disulfide bonds. Eachheavy and light chain contains an amino-terminal variable region ofabout 110 amino acids and constant sequences in the remainder of thechain. The variable region includes several hypervariable regions, orcomplementarity-determining regions (CDRs) that form the antigen-bindingsite of the antibody molecule and determine its specificity. On eitherside of the CDRs of the heavy and light chains is the framework region,a relatively conserved sequence of amino acids that anchors and orientsthe CDRs. The constant region consists of one of five heavy chainsequences (μ, γ, δ, α, or ε) and one of two light chain sequences (κ orλ). The heavy chain constant region sequences determine the isotype ofthe antibody and the effector functions of the molecule.

As used herein, the term “human monoclonal antibody” is intended to meana monoclonal antibody comprising substantially the same heavy and lightchain CDR amino acid sequences as found in a particular humanimmunoglobulin. An amino acid sequence which is substantially the sameas a heavy or light chain CDR exhibits a considerable amount or extentof sequence identity when compared to a reference sequence. Suchidentity is definitively known or recognizable as representing the aminoacid sequence of the particular human monoclonal antibody. Substantiallythe same heavy and light chain CDR amino acid sequence can have, forexample, minor modifications or conservative substitutions of aminoacids. Such a human monoclonal antibody maintains its function ofselectively binding a tumor-specific antigen. The term “human monoclonalantibody” is intended to include a monoclonal antibody withsubstantially human CDR amino acid sequences produced, for example, byrecombinant methods, by lymphocytes or by hybridoma cells.

As used herein, the term “CDR” is intended to mean the non-contiguousantigen combining sites found within the variable region of both heavyand light chain polypeptides. This particular region has been describedby Kabat et al., U.S. Dept. of Health and Human Services, “Sequences ofProteins of Immunological Interest” (1983) and by Chothia et al., J.Mol. Biol. 196:901-917 (1987) and additionally by MacCallum et al., J.Mol. Biol. 262:732-745 (1996), which are incorporated herein byreference, where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orfunctional fragment thereof is intended to be within the scope of theterm as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table I as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody. TABLE I CDR DefinitionsKabat¹ Chothia² MacCallum³ V_(H) CDR1 31-35 26-32 30-35 V_(H) CDR2 50-6552-56 47-58 V_(H) CDR3  95-102  95-102  93-101 V_(L) CDR1 24-34 24-3430-36 V_(L) CDR2 50-56 50-56 46-55 V_(L) CDR3 89-97 89-97 89-96¹Residue numbering follows the nomenclature of Kabat et al., supra²Residue numbering follows the nomenclature of Chothia et al., supra³Residue numbering follows the nomenclature of MacCallum et al., supra

As used herein, the term “functional fragment”, when used in referenceto a human monoclonal antibody, is intended to refer to a portion of themonoclonal antibody which still retains a functional activity. Afunctional activity can be, for example, antigen binding activity orspecificity. A functional activity can also be, for example, an effectorfunction provided by an antibody constant region. Human monoclonalantibody functional fragments include, for example, individual heavy orlight chains and fragments thereof, such as VL, VH and Fd; monovalentfragments, such as Fv, Fab, and Fab′; bivalent fragments such asF(ab′)₂; single chain Fv (scFv); and Fc fragments. Such terms aredescribed in, for example, Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biologyand Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.),New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics,22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178:497-515(1989) and in Day, E. D., Advanced Immunochemistry, Second Ed.,Wiley-Liss, Inc., New York, N.Y. (1990), which are incorporated hereinby reference. The term functional fragment is intended to include, forexample, fragments produced by protease digestion or reduction of ahuman monoclonal antibody and by recombinant DNA methods known to thoseskilled in the art.

The term “VL fragment,” as used herein, refers to a fragment of thelight chain of a human monoclonal antibody which includes all or part ofthe light chain variable region, including the CDRs. A VL fragment canfurther include light chain constant region sequences.

The term “Fd fragment,” as used herein, refers to a fragment of theheavy chain of a human monoclonal antibody which includes all or part ofthe heavy chain variable region, including the CDRs. An Fd fragment canfurther include heavy chain constant region sequences.

The term “Fv fragment,” as used herein, refers to a monovalentantigen-binding fragment of a human monoclonal antibody, including allor part of the variable regions of the heavy and light chains, andabsent of the constant regions of the heavy and light chains. Thevariable regions of the heavy and light chains include, for example, theCDRs. For example, an Fv fragment includes all or part of the aminoterminal variable region of about 110 amino acids of both the heavy andlight chains.

The term “Fab fragment,” as used herein, refers to a monovalentantigen-binding fragment of a human monoclonal antibody that is largerthan an Fv fragment. For example, an Fab fragment includes the variableregions, and all or part of the first constant domain of the heavy andlight chains. Thus, a Fab fragment additionally includes, for example,amino acid residues from about 110 to about 220 of the heavy and lightchains.

The term “Fab′ fragment,” as used herein, refers to a monovalentantigen-binding fragment of a human monoclonal antibody that is largerthan a Fab fragment. For example, a Fab′ fragment includes all of thelight chain, all of the variable region of the heavy chain, and all orpart of the first and second constant domains of the heavy chain. Forexample, a Fab′ fragment can additionally include some or all of aminoacid residues 220 to 330 of the heavy chain.

The term “F(ab′)₂ fragment,” as used herein, refers to a bivalentantigen-binding fragment of a human monoclonal antibody. A F(ab′)₂fragment includes, for example, all or part of the variable regions oftwo heavy chains and two light chains, and can further include all orpart of the first constant domains of two heavy chains and two lightchains.

One skilled in the art knows that the exact boundaries of a fragment ofa human monoclonal antibody are not important, so long as the fragmentmaintains a functional activity. Using well-known recombinant methods,one skilled in the art can engineer a nucleic acid to express afunctional fragment with any endpoints desired for a particularapplication.

As used herein, the term “label” is intended to mean a moiety that canbe attached to a human monoclonal antibody or other molecule of theinvention. Moieties can be used, for example, for therapeutic ordiagnostic procedures.

Therapeutic labels include, for example, moieties that can be attachedto a molecule of the invention and used to reduce the uncontrolledproliferation or viability of a neoplastic cell. A label which candecrease cell proliferation or viability can be, for example, acytotoxic or cytostatic agent, growth factor, cell death receptoragonist or immune modulator.

Diagnostic labels include, for example, moieties which can be detectedby analytical methods. Analytical methods include, for example,qualitative and quantitative procedures. Qualitative analytical methodsinclude, for example, immunohistochemistry and indirectimmunofluorescence. Quantitative analytical methods include, forexample, immunoaffinity procedures such as radioimmunoassay, ELISA orFacs analysis. Analytical methods also include both in vitro and in vivoimaging procedures. Specific examples of diagnostic labels that can bedetected by analytical means include enzymes, radioisotopes,fluorochromes, chemiluminescent markers, and biotin.

A label can be attached directly to a molecule of the invention, or beattached to a secondary binding agent that specifically binds a moleculeof the invention. Such a secondary binding agent can be, for example, asecondary antibody. A secondary antibody can be either polyclonal ormonoclonal, and of human, rodent or chimeric origin.

As used herein, the term “cytotoxic or cytostatic agent” is intended tomean an agent which reduces the viability or proliferative potential ofa cell. Such agents can be attached, for example, to a human monoclonalantibody or other molecule of the invention and used to target cells ortissues. The targeted cells and tissues can include, for example,neoplastic cells and tumors. Examples of targeted cells and tissuesinclude those derived from breast, lung and ovarian tissue. Cytotoxic orcytostatic agents can function in a variety of ways to reduce cellviability or proliferation. Such functions include, for example,inhibiting DNA synthesis, inhibiting cell division, inducing apoptosis,or inducing non-apoptotic cell killing. Specific examples of cytotoxicand cytostatic agents include pokeweed antiviral protein, abrin, ricinand each of their A chains, doxorubicin, cisplastin, Iodine-131,Yttrium-90, Rhenium-188, Bismuth-212, Taxol, 5-Fluorouracil VP-16,Bleomycin, methotrexate, vindesine, adriamycin, vincristine,vinblastine, BCNU, mitomycin and cyclophosphamide and certain cytokinessuch as TNF-α and TNF-β. Thus, cytotoxic or cytostatic agents caninclude, for example, radionuclides, chemotherapeutic drugs, proteinsand lectins.

As used herein, the term “specific binding” is intended to mean aselective interaction of a human monoclonal antibody or functionalfragment thereof with an antigen. For such an interaction to beselective, a human monoclonal antibody will not substantially bind, orcan be made to not substantially bind, to markers other than theparticular antigen. Specific binding can include, for example, bindingaffinity constants of about 10⁵ mol⁻¹ to about 10¹² mol⁻¹. Specificbinding can also include, for example, high avidity interactions.

As used herein, the term “cancer” refers to a pathological conditioncharacterized by the presence of neoplastic cells. Neoplastic cells arecells that exhibit an abnormal morphological or proliferative phenotype.Such cells are characterized by, for example, anchorage independent cellgrowth, proliferation in reduced-serum medium, and loss of contactinhibition. Such cells are also characterized by, for example, abnormalnew growth of tissue, such as a tumor, angiogenic vasculature, andinvasion into surrounding tissue. Neoplastic cells can also metastasizefrom a primary tumor to other sites in the body. For example, a tumor ofthe breast, lung or ovary can metastasize to other organs yet still berecognizable as being comprised of breast, lung or ovarian cells. Thus,the term “tumor” or “cancer” in reference to breast, lung or ovary isintended to include metastases of these tumors to other organs of thebody.

As used herein, the term “effective amount” is intended to mean theamount of a molecule of the invention which can reduce proliferation ofneoplastic cells. The actual amount considered to be an effective amountfor a particular application can depend, for example, on such factors asthe affinity, avidity, stability, bioavailability or selectivity of themolecule, the moiety attached to the molecule, the pharmaceuticalcarrier and the route of administration. Effective amounts can bedetermined or extrapolated using methods known to those skilled in theart. Such methods include, for example, in vitro assays with culturedcells or tissue biopsies and credible animal models.

The invention provides a human monoclonal antibody or functionalfragment having at least one CDR with substantially the amino acidsequence of a CDR of SEQ ID NO:2 or SEQ ID NO:4. The invention alsoprovides a human monoclonal antibody or functional fragment having atleast one CDR with substantially the amino acid sequence of a CDR of SEQID NO:6 or SEQ ID NO:8. The invention further provides human monoclonalantibodies or functional fragments thereof produced by the hybridomacell lines H1140, H2420 and H935. The hybridoma cell lines H1140, H2420and H935 are also provided.

The human monoclonal antibodies produced by the hybridoma cell linesLH11238, LH13, H1140, H2420 and H935 all exhibit specific binding toneoplastic cells as compared to normal cells and, therefore, aretumor-specific human monoclonal antibodies. In particular, the humanmonoclonal antibodies of the invention all selectively bind breastcarcinoma cells and show relatively little binding to normalfibroblasts. For example, the LH11238 antibody specifically binds to anantigen present on the surface and lysosomal compartments of breast andovarian carcinoma cells, as compared to normal fibroblasts, peripheralblood lymphocytes, melanoma cells or lung carcinoma cells. The LH13antibody specifically binds a product produced by breast, lung andovarian carcinoma cells, as compared to normal fibroblasts and melanomacells.

The human monoclonal antibodies produced by hybridoma lines H1140,H2420, H935 and LH13 are of the IgM isotype and λ light chain class,whereas human monoclonal antibodies produced by hybridoma line LH11238is of the IgM isotype and κ light chain class. Further properties of thetumor-specific human monoclonal antibodies are described below in theExamples.

The nucleotide sequence encoding the heavy chain variable region (VH) ofthe human monoclonal antibody produced by LH11238 cell line has beendetermined and is designated SEQ ID NO:1. The VH amino acid sequence ofthe human monoclonal antibody produced by LH11238 cell line isdesignated SEQ ID NO:2. The nucleotide sequence encoding the light chainvariable region (VL) of the human monoclonal antibody produced byLH11238 cell line has also been determined and is designated SEQ IDNO:3. The VL amino acid sequence of the human monoclonal antibodyproduced by LH11238 cell line is designated SEQ ID NO:4.

The nucleotide sequence encoding the heavy chain variable region (VH) ofthe human monoclonal antibody produced by LH13 cell line has beendetermined and is designated SEQ ID NO:5. The VH amino acid sequence ofthe human monoclonal antibody produced by LH13 cell line is designatedSEQ ID NO:6. The nucleotide sequence encoding the light chain variableregion (VL) of the human monoclonal antibody produced by LH13 cell linehas also been determined and is designated SEQ ID NO:7. The VL aminoacid sequence of the human monoclonal antibody produced by LH13 cellline is designated SEQ ID NO:8.

The hybridomas producing the tumor-specific human monoclonal antibodiesof the invention were generated by in vitro immunization of human spleencell cultures with breast carcinoma cells. Briefly, a mixed lymphocytereaction (MLR) was established by co-culturing single cell suspensionsisolated from allogeneic human spleens. Tumor-reactive lymphocytes weresubsequently enriched by incubating MLR cultures either with monolayersor with enriched plasma membranes of breast carcinoma cells. In order toprovide a permanent source of human monoclonal antibodies of theinvention, immunized lymphocytes were immortalized either by fusion withthe K6H6/B5 heteromyeloma cell line or by transformation with EBVfollowed by fusion with K6H6/B5 cells. The particular source of antigenand the immortalization procedures used to generate each of thehybridoma cell lines of the invention are described more fully below inthe Examples.

The tumor-specific human monoclonal antibodies of the invention can alsobe generated by methods known to those skilled in the art. These methodsinclude, for example, in vivo and in vitro enrichment of tumor-reactivelymphocytes. For example, an individual with a breast, lung or ovariantumor can possess lymphocytes which express antibodies that specificallybind tumor-specific antigens, including, for example, LH13, LH11238,H1140, H2420 or H935 antigens. Such lymphocytes can be isolated, forexample, from the peripheral blood or from the spleen of the patient,and immortalized as described below.

Methods are also well known in the art for in vitro enrichment oftumor-reactive human lymphocytes, using tumor-specific antigens. Thesource of antigen can be, for example, substantially pure antigen, tumorcells or tumor cell fractions. A substantially pure antigen can beprepared by any of the methods well known to those skilled in the art,including, for example, chromatography, electrophoretic separation andimmuno-isolation.

An antigen useful for preparing human monoclonal antibodies of theinvention can be, for example, neoplastic cells. The neoplastic cellscan be, for example, cells obtained directly from tumors, culturedprimary tumor cells or established cell lines. Such cells can originatefrom any organ, tissue or fluid of the body, including, for example,breast, lung or ovary. The cancer cells can be untreated, fixed orgrowth-arrested. The fixation can be by any number of methods known tothose skilled in the art, including, for example, chemical fixation.Useful chemicals for fixation include, for example, paraformaldehyde,glutaraldehyde, methanol, or acetone. The cells can alternatively begrowth-arrested using cytostatic agents. A specific example of acytostatic agent is mitomycin C.

An antigen useful for preparing human monoclonal antibodies of theinvention can can also be a fraction of tumor cells. The tumor cellfraction can be, for example, cellular membranes, cytoplasmic contents,or nuclei. Methods for cell fractionation are well known in the art. Anantigen for preparing monoclonal antibodies of the invention can also bean antigen secreted by tumor cells. Such an antigen can be prepared, forexample, by isolation of conditioned medium or cell matrix of tumorcells using procedures known in the art.

An antigen prepared as described above can be used to stimulate humanlymphocytes to generate the tumor-specific human monoclonal antibodiesof the invention. Human lymphocytes can be obtained, for example, fromthe peripheral blood of live individuals, or from the spleen ofindividuals who are deceased or undergoing surgery. Lymphocytes can becultured with antigen directly. Alternatively, lymphocytes can becultured with antigen in a mixed lymphocyte reaction in the presence ofallogeneic lymphocytes. Appropriate culturing conditions for aparticular antigen and lymphocyte source can readily be determined bythose skilled in the art.

If desired, antigen-primed lymphocytes enriched by in vivo or in vitrostimulation as described above can be immortalized by any of a number ofprocedures known to those skilled in the art. Immortalization provides apermanent source of tumor-specific human monoclonal antibodies.Immortalization of lymphocytes can be accomplished by, for example,fusion with an immortal cell line. Such immortal cell lines useful forcell fusion can be, for example, human myeloma cells or humanlymphoblastoid B cell lines. Fusion partners can also be rodent myelomacells or human:rodent heteromyeloma cell lines. The heteromyeloma cellline can be, for example, the human:mouse heterohybridoma cell lineK6H6/B5. Alternatively, antigen-primed lymphocytes can be immortalizedby viral transformation, using, for example, viruses. A useful virus forimmortalization by viral transformation is EBV. Antigen-primedlymphocytes can also be immortalized by viral transformation followed byfusion. Viral transformation followed by fusion can be, for example, EBVtransformation followed by fusion with the K6H6/B5 cell line. Cultureconditions for lymphocyte fusion or viral transformation can readily bedetermined by those skilled in the art.

Immortalized lymphocytes can be screened for the production of humanmonoclonal antibodies that specifically bind to human tumor cells usingimmunoassays known to those skilled in the art. Such immunoassaysinclude, for example, quantitative and qualitative immunoassays.Qualitative immunoassays include, for example, precipitin methods,agglutinin reactions, immunohistochemistry, immunofluorescence,immunoblotting and immunoprecipitation. Quantitative immunoassaysinclude, for example, immunoaffinity methods such as radioimmunoassay,Facs analysis, and ELISA analysis. ELISA analysis can be direct,sandwich or competitive. Immunoassays can be direct, using, for example,a labeled human monoclonal antibody. Such methods can alternatively beindirect, using, for example, a labeled anti-human secondary antibody.The label can be, for example., a fluorescent label, an enzyme, aradioisotope, or biotin. Detection can be by spectrophotometric,radiographic or chemiluminescent means, depending on the immunoassay.

The tumor-specific antigen sample used for screening monoclonalantibodies for tumor reactivity need not be the same as the antigensample used to immunize the human lymphocytes. The antigen used inscreening can be, for example, substantially purified antigen, live orfixed tumor cell monolayers, live or fixed tumor cell suspensions,fractions of tumor cells, or sections of tumor biopsies, depending onthe assay procedure employed. The tumor cells can be, for example, humanbreast, ovarian or lung carcinoma cells.

The tumor-specific human monoclonal antibodies of the invention do notbind, bind only minimally, or can be made to bind only minimally tonormal cell antigens. Normal cells or fractions of normal cells can beused as controls for screening human monoclonal antibodies. Such normalcells can be, for example, live or fixed normal tissues or cultured celllines. Cultured normal cell lines that can be used as controls include,for example, human fibroblasts and peripheral blood lymphocytes.Although any normal cell can be used as a control, the selection of aparticular control will be based, in part, on the specificity of theparticular tumor-specific monoclonal antibody which is being screened.For example, if a human tumor-specific monoclonal antibody is producedand screened against a carcinoma cell, then one type of normal cellwhich can be used as a comparison is a normal epithelial cell culture orcell line. Similarly, if a human tumor-specific monoclonal antibody isproduced and screened against a sarcoma cell, then one type of normalcell which can be used as a comparison is a normal fibroblast cellculture or cell line. A normal cell culture or cell line from the sametissue type or from the same individual can also provide a normalcontrol. Those skilled in the art will know what is an appropriate typeof normal cell to be used as a control to determine specific binding toa particular type of tumor cell.

Tumor-specific human monoclonal antibodies can be purified andquantitated for use in immunodiagnostic and immunotherapeuticprocedures. Such purification methods are well known to those skilled inthe art and depend on the source of human monoclonal antibodies and theparticular application. Purification methods can include, for example,precipitation, electophoresis, chromatography, and immunoaffinitypurification. The purified antibodies can be quantitated in comparisonwith known standard controls, using, for example, spectrophotometry orimmunoassays known in the art.

To further characterize tumor-specific human monoclonal antibodies,their class and subclass can also be determined by immunoassays thatmeasure the presence of individual heavy and light chain polypeptides.Such immunoassays include ELISA assays and are known to those skilled inthe art.

The invention further provides functional fragments of thetumor-specific human monoclonal antibodies of the invention. Afunctional fragment of a human monoclonal antibodies maintains abiological activity, such as specific binding or an effector function. Afunctional fragment can therefore be beneficially used for the detectionand treatment of cancer.

Functional fragments include fragments with substantially the same heavyand light chain variable regions as a tumor-specific human monoclonalantibody of the invention. For example, functional fragments includefragments wherein at least one of the CDR sequences consist ofsubstantially the same amino acid sequence as the CDR sequences of atumor-specific human monoclonal antibody of the invention. Suchfunctional fragments include, for example, VL, Fd, Fv, Fab, Fab′,F(ab′)₂ and Fc fragments. For example, a functional fragment could haveone or more of the three CDR sequences of the VL, or one or more of thethree CDR sequences of the VH, or a combination of VL and VH CDRs of ahuman monoclonal antibody of the invention. The appropriate number andcombination of VH and VL CDR sequences can be determined by thoseskilled in the art depending on the desired affinity and specificity andthe intended use of the functional fragment.

Functional fragments of human monoclonal antibodies of the invention canreadily be produced and isolated using methods well known to thoseskilled in the art. Such methods include, for example, proteolyticmethods, recombinant methods and chemical synthesis. Proteolytic methodsfor the isolation of functional fragments use human monoclonalantibodies as starting material. Enzymes suitable for proteolysis ofhuman monoclonal antibodies include, for example, papain, pepsin andelastin. The appropriate enzyme can be readily chosen by one skilled inthe art, depending on, for example, whether monovalent or bivalentfragments are required. For example, papain cleavage results in twomonovalent Fab′ fragments that bind antigen and an Fc fragment. Pepsincleavage, for example, results in a bivalent F(ab′)₂ fragment. A F(ab′)₂fragment of the invention can further be reduced using, for example, DTTor β-mercaptoethanol to produce two monovalent Fab′ fragments.

Functional fragments produced by proteolysis can be purified by affinityand column chromatographic procedures. For example, undigestedantibodies and Fc fragments can often be removed by binding to proteinA. Additionally, functional fragments can be purified by virtue of theircharge and size, using, for example, ion-exchange and gel filtrationchromatography. Such methods are well known to those skilled in the art.

Recombinant methods for producing functional fragments of humanmonoclonal antibodies begin with the isolated nucleic acid of desiredregions of the immunoglobulin heavy and light chains. Such regions caninclude, for example, all or part of the variable region of the heavyand light chains. Such regions can particularly include the CDRs of theheavy and light chains.

The invention provides an isolated nucleic acid encoding a humanmonoclonal antibody or functional fragment thereof, comprising anucleotide sequence encoding substantially the amino acid sequence of atleast one CDR encoded by SEQ ID NO:1 or SEQ ID NO:3. Also provided is anisolated nucleic acid encoding a CDR, comprising substantially the aminoacid sequence of a CDR encoding by SEQ ID NO:1 or SEQ ID NO:3. Theinvention further provides an isolated nucleic acid encoding a humanmonoclonal antibody or functional fragment thereof, comprising anucleotide sequence encoding substantially the amino acid sequence of atleast one CDR encoded by SEQ ID NO:5 or SEQ ID NO:7. Also provided is anisolated nucleic acid encoding a CDR, comprising substantially the aminoacid sequence of a CDR encoding by SEQ ID NO:5 or SEQ ID NO:7.

A nucleic acid encoding a human monoclonal antibody or functionalfragment of the invention can be produced using methods known to thoseskilled in the art. One useful procedure for isolating such DNA beginswith cDNA which can be reverse-transcribed from RNA of hybridoma cellsthat produce a tumor-specific human monoclonal antibody. Methods forcDNA synthesis are well known in the art. A cDNA encoding a functionalfragment of a heavy or light chain can be amplified using, for example,the polymerase chain reaction (PCR). The PCR technology is the subjectmatter of U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202,all of which are incorporated by reference herein. Suitable primers forPCR are known and can be determined by those of skill in the art usingconserved sequences which flank the particular functional fragment of aheavy or light chain. Suitable PCR conditions are known and can alsosimilarly be determined by those skilled in the art.

A nucleic acid encoding a functional fragment of a human monoclonalantibody of the invention can also be directly synthesized by methods ofoligonucleotide synthesis known in the art. Alternatively, smallerfragments can be synthesized and joined to form a larger functionalfragment using recombinant methods known in the art.

Nucleic acids encoding a functional fragment of a human monoclonalantibody of the invention can be cloned into a suitable expressionvector and expressed in a suitable host. A suitable vector and host cellsystem can allow, for example, co-expression and assembly of functionalfragments of the heavy and light chains. Suitable systems for theexpression of antibody fragments can be determined by those skilled inthe art and include, for example, M13 phage immunoexpression vectors.Recombinant functional fragments of the invention can be substantiallypurified using methods known in the art, and which depend on theparticular vector and host expression system used.

Isolated nucleic acids encoding tumor-specific human monoclonalantibodies or functional fragments can also be engineered to produceantibodies with optimal properties such as affinity, selectivity,avidity, stability or bioavailability. Such modifications can include,for example, addition, deletion, or substitution of amino acid residuesor substitution of a D-amino acid or amino acid mimetic, so long as thefragment maintains a functional activity such as, for example, antigenbinding specificity.

Amino acid substitutions can be introduced by mutating nucleic acidcodons encoding the particular amino acid using methods known in theart. Single or multiple codons can be varied, so long as the fragmentretains a functional activity. Rapid methods for making and screeningmultiple simultaneous changes are well known within the art and can beused to produce a library of encoding nucleic acids which contain allpossible or all desired changes and then expressing and screening thelibrary for human monoclonal antibodies or fragments which retainfunction. Such methods include, for example, codon based mutagenesis,synthesis of stochastic oligonucleotides and partially degenerateoligonucleotide synthesis.

Codon based mutagenesis is the subject matter of U.S. Pat. Nos.5,264,563 and 5,523,388 and is advantageous for the above proceduressince it allows for the production of essentially any and all desiredfrequencies of encoded amino acid residues at any and all particularcodon positions within an oligonucleotide. Such desired frequenciesinclude, for example, the truly random incorporation of all twenty aminoacids or a specified subset thereof as well as the incorporation of apredetermined bias of one or more particular amino acids so as toincorporate a higher or lower frequency of the biased residues comparedto other incorporated amino acid residues.

Synthesis of stochastic oligonucleotides and partially degenerateoligonucleotide synthesis can similarly be used for producing andscreening for functionally equivalent amino acid changes. However, dueto the degeneracy of the genetic code, such methods can incorporateredundancies at a desired amino acid position (see, for example, U.S.Pat. No. 5,723,323). Stochastic oligonucleotide synthesis includes thecoupling of all four nucleotides at each nucleotide position within acodon. Other stochastic methods of synthesis also exist which can resultin degenerate or partially degenerate oligonucleotides oroligonucleotides which encode completely random amino acid sequences(see, for example, U.S. Pat. No. 5,723,323).

Partially degenerate oligonucleotide synthesis is the coupling of equalportions of all four nucleotides at the first two nucleotide positions,for example, and equal portions of two nucleotides at the thirdposition. Both of these latter synthesis methods can be found describedin, for example, Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382,(1990) and Devlin et al., Science 249:404-406, (1990).

For certain therapeutic and diagnostic applications it may be preferableto use antibodies with the same antigen specificity but with differentisotypic or allotypic determinants. Such antibodies could have, forexample, decreased immunogenicity, increased stability, or more optimaleffector functions. Thus, functional fragments of the invention caninclude those obtained by cloning the CDR sequences of a humanmonoclonal antibody of the invention into different framework regions.Such different framework regions can be obtained from different species,different human individuals, or different heavy or light chain classesfrom the same or different individual. Such CDR grafting methods arewell known to those skilled in the art.

Functional activity of fragments of the invention can be evaluated by,for example, methods described above for determining theimmunoreactivity of human monoclonal antibodies. Particularly usefulmethods for determining functional activity of fragments includecompetitive radioimmunoassay and competitive ELISA assay.

The present invention provides pharmaceutical compositions containing ahuman monoclonal antibody or functional fragment of the invention and apharmaceutical carrier. Such compositions can be used to administer ahuman monoclonal antibody or fragment to reduce the proliferation orviability of neoplastic cells. Such compositions can also be used todetect neoplastic cells.

Suitable pharmaceutical carriers for the methods of the invention arewell known and include, for example, aqueous solutions such asphysiologically buffered saline, and other solvents or vehicles such asglycols, glycerol, oils or injectable organic esters. A pharmaceuticalcarrier can contain a physiologically acceptable compound that acts, forexample, to stabilize or increase the solubility of a pharmaceuticalcomposition. Such a physiologically acceptable compound can be, forexample, a carbohydrate, such as glucose, sucrose or dextrans; anantioxidant, such as ascorbic acid or glutathione; a chelating agent; alow molecular weight protein; or another stabilizer or excipient.Pharmaceutical carriers, including stabilizers and preservatives, aredescribed, for example, in Martin, Remington's Pharm. Sci., 15th Ed.(Mack Publ. Co., Easton, 1975), which is incorporated herein byreference.

Those skilled in the art will know that the choice of the pharmaceuticalmedium and the appropriate preparation of the composition will depend onthe intended use and mode of administration.

The invention also provides a CDR or functional fragment thereof havingsubstantially the amino acid sequence of a CDR of SEQ ID NO:2 or SEQ IDNO:4. The invention also provides a CDR or functional fragment thereof,having substantially the amino acid sequence of a CDR of SEQ ID NO:6 orSEQ ID NO:8.

A CDR or functional fragment thereof of the invention can be produced bymethods known in the art and as described above. For example, a CDR ofthe invention can be produced by recombinant means or chemicalsynthesis. A CDR of the invention can be advantageously used, forexample, to generate anti-idiotypic antibodies that selectively bind thetumor-specific human monoclonal antibodies of the invention. Methods forproducing and using anti-idiotypic antibodies for diagnostic andtherapeutic purposes are well known in the art.

The invention further provides a method of reducing neoplastic cellproliferation by administering to the cell an effective amount of ahuman monoclonal antibody or functional fragment produced by a cell lineselected from the group consisting of H1140, H2420 and H935. Alsoprovided is a method of reducing neoplastic cell prolieration byadministering to the cell an effective amount of a human monoclonalantibody or functional fragment having at least one CDR withsubstantially the amino acid sequence of a CDR of SEQ ID NO:2 or SEQ IDNO:4. Further provided is a method of reducing neoplastic cellproliferation by administering to the cell an effective amount of ahuman monoclonal antibody or functional fragment having at least one CDRwith substantially the amino acid sequence of a CDR of SEQ ID NO:6 orSEQ ID NO:8. A human monoclonal antibody or functional fragment used forreducing neoplastic cell proliferation can further include a label, suchas a cytotoxic or cytostatic agent, and can be combined with apharmaceutical carrier.

An effective amount of a molecule of the invention for reducingneoplastic cell proliferation is known or can readily be determined byone skilled in the art using in vitro methods or credible animal models.In vitro methods can include, for example, determining an effectiveamount of a composition for reducing neoplastic cell growth orneoplastic cell metastasis. A neoplastic cell used in an in vitro methodfor assaying reduction in growth or metastasis can be, for example, atumor cell line or an ex vivo culture of a tumor. The cell line or tumorcan be, for example, of breast, lung or ovarian tissue in origin. Aneffective amount for inhibiting neoplastic cell growth can be, forexample, an effective amount for inhibiting DNA synthesis, inhibitingcell division, inducing apoptosis, inducing non-apoptotic killing, orinhibiting angiogenesis. An effective amount for inhibiting metastasisof a neoplastic cell can be, for example, an amount effective forinhibiting cell motility, cell migration, cell attachment, cell invasionor cell proliferation.

An effective amount of a molecule of the invention for reducingneoplastic cell proliferation can also be determined from xenografts ofhuman tumors in rodents. The rodent can be, for example, a rat or amouse. The mouse can be, for example, normal or immunocompromised. Animmunocompromised mouse can be, for example, a nude mouse or a scidmouse. Such species are well known in the art and can be obtained fromcommercial sources. Human cancer cells can be introduced into an animalby a number of routes, including subcutaneously, intraveneously andintraperitoneally. Following establishment of a tumor, the animals canbe treated with different doses of a molecule of the invention, andtumor mass or volume can be determined. Efficacy can be measured aspartial or complete regression of the tumor. An effective dose fortreating cancer results in more partial and complete regressions oftumors.

An effective amount of a molecule of the invention can be determined byone skilled in the art and will depend on such factors as age, bodyweight, sex and medical condition of the individual, and the particularroute of administration of the therapeutic agent. Useful routes ofadministration of a composition of the invention for treating cancerinclude, for example, intramuscular, intratumoral, intravascular,intraperitoneal, subcutaneous or intranasal routes.

The efficacy of a particular treatment in cancer patients can bedetermined by one skilled in the art. For example, in vivo or in vitrodiagnostic methods, such as those described below, can be used todetermine that a tumor has regressed or been eliminated followingtreatment. Additionally, normal prognostic indicators, such as survivaland increased quality of life for the cancer patient, can be used.

The invention provides a method of detecting neoplastic cells bycontacting a sample with a human monoclonal antibody or functionalfragment produced by a cell line selected from the group consisting ofH1140, H2420 and H935, and detecting the specific binding of the humanmonoclonal antibody or functional fragment to the sample, wherein thepresence or increased level compared to a normal cell of the humanmonoclonal antibody or functional fragment indicates the presence of orpredisposition to cancer. The invention also provides a method ofdetecting neoplastic cells by contacting a sample with a humanmonoclonal antibody or functional fragment having substantially theamino acid sequence of a CDR of SEQ ID NO:2 or SEQ ID NO:4, anddetecting the specific binding of the human monoclonal antibody orfunctional fragment to the sample, wherein the presence or increasedlevel compared to a normal cell of the human monoclonal antibody orfunctional fragment indicates the presence of or predisposition tocancer. The invention further provides a method of detecting neoplasticcells by contacting a sample with a human monoclonal antibody orfunctional fragment having substantially the amino acid sequence of aCDR of SEQ ID NO:6 or SEQ ID NO:8, and detecting the specific binding ofthe human monoclonal antibody or functional fragment to the sample,wherein the presence or increased level compared to a normal cell of thehuman monoclonal antibody or functional fragment indicates the presenceof or predisposition to cancer.

As used herein, the term “sample” is intended to mean any biologicalfluid, cell, tissue, organ or portion thereof, that includes orpotentially includes neoplastic cells. A biological fluid can be, forexample, blood or lymph. A tissue can be, for example, breast, ovary, orlung. The sample can be an in vivo or in vitro sample. An in vitrosample can be, for example, a histologic section, a specimen obtained bybiopsy, or cells that are placed in or adapted to tissue culture. Asample can be prepared by methods known in the art suitable for theparticular format of the detection method.

A sample can be contacted with a human monoclonal antibody or functionalfragment of the invention and specific binding of the human monoclonalantibody to the sample can be detected. Such contacting can be in vivoor in vitro, as determined by one skilled in the art depending on theformat of the detection method used. Specific binding of the humanmonoclonal antibody to the sample can be determined by immunoassays asdescribed above. Such immunoassays include, for example, both in vivoand in vitro immunoassays. In vivo immunoassays include, for example,radioimaging. Such a method involves contacting a sample within anindividual with a monoclonal antibody of the invention, and detectingspecific binding by, for example, radiographic imaging. In vitroimmunoassays include both qualitative and quantitative assays, such as,for example, immunohistochemistry, immunofluorescence, radioimmunoassay,Facs analysis, immunoblotting, immunoprecipitation and ELISA analysis,as described above.

The determination that neoplastic cells are present can be made bydetermining that a specifically bound human monoclonal antibody of theinvention is present or is at an increased level compared to a normalcell. As described above, one skilled in the art would be able todetermine an appropriate normal cell to use for comparison with aparticular type of neoplastic cell.

The invention further provides a substantially pure human tumor-specificantigen. The term “tumor-specific antigen” is intended to mean anantigen which is preferentially expressed by human tumor cells. The term“preferentially expressed by human tumor cells” is intended to mean thata tumor-specific antigen is expressed by human tumor cells and isexpressed at a substantially lower level by normal human cells. Suchtumor cells that express a tumor-specific antigen of the invention canbe, for example, of breast, lung or ovarian tissue origin.

A human tumor-specific antigen of the invention is specifically reactivewith a human monoclonal antibody of the invention. Such an antigen canspecifically react with, for example, a human monoclonal antibody orfunctional fragment produced by LH11238, LH13, H1140, H2420 or H935hybridoma cell lines. Such an antigen can also specifically react with ahuman monoclonal antibody or functional fragment having at least one CDRwith substantially the amino acid sequence of a CDR of SEQ ID NO:2 orSEQ ID NO:4, or with a human monoclonal antibody or functional fragmenthaving at least one CDR with substantially the amino acid sequence of aCDR of SEQ ID NO:6 or SEQ ID NO:8. The antigen reactive with LH11238human monoclonal antibody is a protein present on the cell surface andin lysosomal compartments of breast and ovarian cancer cells as comparedto normal human fibroblasts, peripheral blood lymphocytes, melanomacells or lung carcinoma cells. The antigen reactive with LH13 humanmonoclonal antibody is a secreted glycoprotein produced by breast, lungand ovarian cancer cells as compared to normal fibroblasts or melanomacells. Further properties of LH11238 and LH13 antigens are described inthe Examples.

Human tumor-specific antigens of the invention can beneficially be usedfor the treatment and diagnosis of cancer. For example, such antigenscan be used to generate additional binding agents that specifically bindthe human tumor-specific antigen for use in therapeutic and diagnosticprocedures. Such binding agents can, for example, inhibit or stimulatethe function of a tumor-specific antigen, or modulate the immune system,such that a neoplastic cell is growth-arrested or killed. Such bindingagents can also be conjugated to a label, such as, for example, acytotoxic or cytostatic agent, that causes the death or arrest of thetumor cells. Useful agents that bind to a tumor-specific antigen of theinvention include, for example, ligands, receptor antagonists andantibodies.

A substantially pure tumor-specific antigen can also be used in thetreatment of cancer by vaccinating a patient having cancer, or at riskof developing cancer with an effective amount of the antigen. Followingvaccination, the immune system of the individual will be able toprevent, reduce the proliferation of, or eliminate neoplastic cellsexpressing such an antigen.

A substantially pure tumor-specific antigen of the invention can also bebeneficially used in methods for detecting binding of a tumor-specificbinding agent, such as a human monoclonal antibody or functionalfragment of the invention. For example, such an antigen can be used inan immunoassay such as competitive ELISA.

An appropriate starting material for isolating a substantially pureantigen of the invention can be identified by, for example, screening apanel of human tumor cells, using immunoaffinity procedures well knownin the art. For example, as described in Example II, ELISA analysis canbe used to determine a useful cell source of antigen for subsequentpurification. A useful cell source of antigen can be, for example,breast, ovarian, or lung carcinoma. A particularly useful startingmaterial for the purification of a tumor-specific antigen is the H3396breast carcinoma cell line.

An appropriate method for isolating a substantially pure antigen dependson the cellular localization of the antigen. For example, an antigen ofthe invention can be predominantly expressed in the secreted medium,cell surface membrane, vesicular membranes, cytoplasm, or nucleus of theneoplastic cell. Cellular localization of an antigen can be determinedby, for example, immunoassays well known in the art. For example, asdescribed in Examples IV and VI, indirect immunofluorescence and ELISAanalysis can be used to establish the predominant localization of anantigen.

Purification of an antigen can be monitored by immunoaffinity proceduresknown in the art. Particularly useful methods of monitoring purificationinclude, for example, ELISA analysis and immunoblotting.

Tumor-specific antigens can be purified from a particular source bybiochemical procedures well known in the art. For example, purificationcan include centrifugation, chromatographic methods, electrophoreticmethods and immunoaffinity methods, and can be chosen by one skilled inthe art depending on the characteristics of a particular antigen.Centrifugation procedures can be used to concentrate or enrich for asubcellular fraction which contains an abundant amount of the antigen.Such subcellular fractionation procedures are well known in the art.Chromatographic methods are also well known in the art, and includemethods of separating an antigen from contaminants based on its size ordifferential affinity for particular resins. Such resins can include,for example, size exclusion resins, ion exchange resins, and lectincolumns. As described in Example VI, a particularly useful resin forpurification of an antigen of the invention is Q SEPHAROSE FAST FLOWagarose resin. Electrophoretic methods are also well known in the art,and include one and two-dimensional electrophoresis through, forexample, acrylamide or agarose gels. Immunoaffinity procedures are alsowell known in the art and include compounds conjugated to a humanmonoclonal antibody of the invention. Useful compounds for conjugatingan antibody for immunoaffinity purification of a tumor-specific antigeninclude chromatographic resins and Protein A.

Thus, following well-known biochemical procedures, one skilled in theart can readily isolate a substantially pure tumor-specific antigen ofthe invention for use in therapeutic and diagnostic procedures.

Substantially purified tumor-specific antigens of the invention can alsobe prepared from nucleic acids encoding tumor-specific antigens of theinvention by recombinant methods known to those skilled in the art.

The invention provides isolated nucleic acids encoding humantumor-specific antigens. Nucleic acids encoding human tumor-specificantigens can be isolated by methods known to those skilled in the art.Such methods include, for example, using monoclonal antibodies of theinvention to screen an expression library. Other methods include, forexample, screening a cDNA or genomic library using degenerateoligonucleotides as hybridization probes. The sequence of such adegenerate oligonucleotide can be determined by microsequencing anisolated tumor-specific antigen of the invention or fragment thereof.

Other methods known to those skilled in the art for producing a nucleicacid of a tumor-specific antigen include, for example, the polymerasechain reaction (PCR), using degenerate oligonucleotide primers obtainedfrom amino acid sequence of a tumor-specific antigen of the invention.Desired sequences can be amplified exponentially starting from as littleas a single gene copy by means of PCR.

The above described methods are known to those skilled in the art andare described, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1992) andthe various references cited therein and in Ansubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989); and in Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York (1989). These references and thepublications cited therein are hereby expressly incorporated byreference.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Production of Tumor-Specific Human Monoclonal Antibodies

This Example shows production of hybridomas that secrete humanmonoclonal antibodies specifically reactive with tumor cells. Aprocedure for immunizing normal lymphocytes in vitro with tumor cells orcell membranes prior to immortalization is described. This procedureallowed enrichment of lymphocytes producing novel tumor-reactive humanmonoclonal antibodies. These human monoclonal antibodies will be usefulfor cancer immunotherapeutic and immunodiagnostic procedures.

Lymphocytes were prepared as follows. Spleen tissue was isolated fromaccident victims, fragmented, and forced through a no. 50 mesh wirescreen (Bellco, Vineland, N.J.). The cells were collected bycentrifugation at 250×g for 10 min and RBC were removed by ammoniumchloride lysis. The remaining cells were washed, resuspended in freezingmedium (40% RPMI, 50% FCS and 10% DMSO) at a concentration of 100 to300×10⁶ cells/ml, frozen in 1.5-ml aliquots, and stored in liquidnitrogen. Both adherent cells and lymphocytes were isolated by thisprocedure.

A mixed lymphocyte reaction (MLR) was then established as follows.Frozen single-cell splenocyte preparations (described above) from twodifferent donors were thawed by gentle shaking at 37° C., washed twicewith RPMI, and collected by centrifugation for 10 min at 250×g. Afterthe second wash, 3×10⁶ splenocytes from each of the sources werecombined in 2 ml of RPMI containing 1.5 mM HEPES, pH 7.4 (FisherScientific), 10% FBS (HyClone), 2 mM L-glutamine, non-essential aminoacids, 1 mM sodium pyruvate, and 100 μg/ml gentamicin sulfate and placedin a 24-well tissue culture dish. This MLR, together with antigenicstimulation (described below), endogenously produced the requiredlymphokines.

MLR cultures were then stimulated with either mitomycin-treated H3396tumor cells, plasma membrane preparations from H3396 cells, orparaformaldehyde-fixed H3922 tumor cells. H3922 and H3396 are culturedcell lines established from metastases of human breast adenocarcinoma,which were explanted and maintained in culture. Each cell line wasderived from a different explant.

Mitomycin-treated H3396 cells were prepared as follows. H3396 cells wereplated in 24-well culture dishes, grown to confluency, and treated for12-15 h with 0.1 μg/ml mitomycin C. This concentration arrested celldivision in the tumor cell lines for approximately seven days. Followingthe 12-15 h incubation, the mitomycin C-containing media was removed andthe cells were washed three times with 2 ml of phosphate buffered saline(PBS).

Paraformaldeyde-fixed H2922 tumor cells were prepared by incubatingcells for 15 min at 25° C. in 2% paraformaldehyde in PBS after removingthe culture medium. The cells were then washed four times with PBS priorto use.

Plasma membranes from H3396 cells were isolated as follows. Tenconfluent 150-mm dishes of H3396 cells were each rinsed twice with 10 mlof ice-cold (Tris-buffered saline) TBS and harvested in 2 ml/150-mm dishof (Tris saline) TS containing 1 μg/ml leupeptin, 1 μg/ml pepstatin, and2 mM AEBSF. The cells were broken in a 15 ml Dounce homogenizer with 40strokes using a type A pestle. The lysate was centrifuged at 800×g at 4°C. for 5 min to remove unbroken cells and nuclei. The supernatant wassaved and the pellet was resuspended in 0.5 volumes of TS buffer,homogenized and centrifuged at 800×g. The supernatant was combined withthe first supernatant and centrifuged at 10,000×g at 4° C. for 2 h. Thesupernatant was discarded and the pellet was resuspended in 2 ml ofwater. A 2 ml Dounce and type B pestle was used to resuspend themembrane pellet. Phase separation of membranes in a 6.4% polymer systemwas performed on ice by mixing 2.56 ml of 20% dextran, 1.28 ml of 40%polyethylene glycol, 0.20 ml of 0.2 M potassium phosphate, pH 7.2, 0.8ml of 1 M sucrose, and 2.16 ml of water. To this mixture 1 ml ofmembranes were added and the tube was inverted 20 times end over end.The phases were separated by centrifugation at 800×g at 4° C. for 5 min.The upper phase was drawn off and mixed with the lower phase recoveredfrom a blank (water) sample. Likewise, the lower phase, including thematerial at the interface, was mixed with the upper phase recovered froma blank sample. Both phases were mixed, inverted 20 times end over end,and separated by centrifugation at 800×g at 4° C. for 5 min. Thematerial recovered from the upper phase of both samples was combined,the volume was adjusted to 21 ml with TBS, and the membranes werecollected by centrifugation at 100,000×g at 4° C. for 2 h. Thesupernatant was discarded and the pellet was resuspended with a 2 mlDounce homogenizer with a B pestle in 1-2 ml of TS buffer containing theprotease inhibitors. The membranes were stored at 4° C. for 12 h, atwhich time the insoluble material was separated from the supernatant bycentrifugation at 800×g at 4° C. for 10 min. The pellet was suspended asecond time in 1-2 ml of TS buffer containing protease inhibitors andstored as a suspension at 4° C. The supernatant is referred to asfraction 1 while the resuspended particulate material is referred to asfraction 2. Plasma membranes in fraction 1 were enriched greater than10-fold as determined by measuring 5′-nucleotidase or phosphodiesteraseactivity. The preparations had minimal succinate-dependent cytochrome Creductase (mitochondria), or NADPH-dependent cytochrome C reductase(endoplasmic reticulum) activity.

MLR cultures were immunized in vitro by incubation for three days withone of the following: monolayers of mitomycin C-treated H3396 cells,monolayers of paraformalehyde-fixed H3922 cells, 5 mg of plasma membranefraction 1 from H3396 cells, or with 10 mg of plasma membrane fraction 2from H3396 cells.

In vitro immunized lymphocytes were immortalized by either of twoalternative methods. In the first method, lymphocytes were fused withK6H6/B5 heteromyeloma cells. K6H6/B5 cells were maintained in spinnerculture in a 1:1 mixture of RPMI and Iscoves's modification of DMEM,supplemented with 10% FCS, 1% nonessential amino acids, 2 mM glutamine,and 1 mM sodium pyruvate. Approximately 4×10⁶ lymphocytes were combinedwith 2×10⁶ log phase K6H6/B5 cells and washed twice with RPMI. One ml ofa mixture of 35% polyethylene glycol (approximate m.w. 1450) and 7.5%DMSO in RPMI was added over 1 min, then gradually diluted with RPMI to 4ml and then diluted with RPMI supplemented with 10% FCS to 16 ml. Thelymphocyte concentration was then adjusted to 5×10⁴ cells/ml, and 0.2 mlof cells were seeded in 96-well cell culture dishes in hybridoma medium(RPMI supplemented with 10% FCS, 1% nonessential amino acids, 2 mMglutamine, 1 mM sodium pyruvate, 15 mM HEPES, pH 7.4 and 0.1 mg/mlgentamycin) containing HAT (13.6 μg/ml hypoxanthine, 3.8 μg/mlthymidine, 1 μg/ml azaserine) and 1.0 μM ouabain, to select againstunfused cells.

Hybridomas were screened for reactivity as described below, and clonesof interest were expanded. Using this one-step immortalization method,10-50 hybridomas were produced per 10⁶ lymphocytes fused; however, only5% of the interesting clones were stable through three rounds of cellculturing. Hybridoma clones H1140, H2420 and H935 were obtained by thisimmortalization procedure.

In an effort to improve clonal stablility, a second, two-step method forimmortalizing in vitro immunized lymphocytes was evaluated, involvingfirst transforming cells with EBV and then fusing clones of interestwith K6H6/B5 heteromyeloma cells. Equal volumes of lymphocytes (8×10⁴cells/ml) and EBV-transformed 1A2/C7 cells (2×10⁴ cells/ml) werecombined and the total volume was doubled by the addition of hybridomamedium. Hypoxanthine, thymidine and azaserine were added such that theirfinal concentrations were 13.6 μg/ml, 3.8 μg/ml, and 1 μg/ml,respectively. Two hundred microliters of the mixture of cells was platedper well in 96-well cell culture dishes to yield a final total of 4000lymphocytes and 1×10⁵ 1A2/C7 cells per well. The cells were fed withhybridoma medium-HAT every three days and assayed for antibodyproduction (described below) after 2 weeks when colonies of cells werevisible.

Transformation of lymphocytes with EBV generated a higher percentage ofantibody-secreting clones than was obtained from fusions of lymphocyteswith the heteromyeloma line (50-100 antibody-secreting clones per 10⁶lymphocytes). However, lymphoblastoid clones generally secreted lowerlevels of antibody (less than 10 μg/ml) and, as was observed with thehybridomas, displayed poor long-term stability (approximately 5% of theinitial clones of interest were stable through three rounds ofexpansion). To address these limitations, tumor-reactive lymphoblastoidclones were fused with K6H6/BS cells as described above, prior tomultiple rounds of expansion.

Formation of hybridomas from lymphoblastoid cells improved the stabilitysuch that 40% of the initial lymphoblastoid clones were stable throughthree rounds of expansion following fusion. The combination of EBVtransformation followed by fusion with a heteromyeloma cell lineresulted in a higher frequency of immortalization of relevantlymphocytes than was achieved utilizing either approach alone. Inaddition, these clones generally secreted greater than 20 μg/ml ofantibody. Hybridoma clones LH11238 and LH13 were produced by thistwo-step immortalization method.

The immunization and immortalization conditions used to producehybridoma cell lines H1140, H2420, H935 LH11238 and LH13 are summarizedin Table 2. TABLE 2 Immunization and immortalization conditions used togenerate hybridoma cell lines producing tumor- specific human monoclonalantibodies. Hybridoma Immunization Immortalization H1140  5 mg H3396plasma Fusion with K6H6/B5 membrane fraction 1 cells H2420  5 mg H3396plasma Fusion with K6H6/B5 membrane fraction 1 cells H935 confluentmonolayer of Fusion with K6H6/B5 mitomycin C-treated cells H3396 cellsLH11238 10 mg H3396 plasma EBV transformation membrane fraction 2 andfusion with K6H6/B5 cells LH13 monolayer of 4 × 10⁴ EBV transformationparaformaldehyde-fixed and fusion with H3922 cells K6H6/B5 cells

Culture supernatants from immortalized lymphocytes were initiallyscreened for reactivity against monolayers of live primary tumor cellsusing an ELISA assay. This ensured that reactive antibodies recognizedsurface antigens, and also avoided artifacts associated with screeningfixed cells. Tumor cells were plated in 96-well cell culture dishes at acell density sufficient to produce 90-95% confluent monolayers 12-24 hlater. Just prior to use the media was removed and 50 μl of supernatantfrom either hybridomas or EBV transformed cells was added to the wellsand incubated at 4° C. for 2 h. Control wells (background) wereincubated with 50 μl of fresh hybridoma media.

Following incubation, supernatants were aspirated and the cells weregently rinsed three times with 200 μl of PBS. The cells were thenincubated for 1-2 h with 50 μl of goat anti-human lg (H+L) alkalinephosphatase conjugate which had been diluted 1000-fold in 1% BSA-PBS.The detection antibody was aspirated and the cells were gently rinsedfour times as described above. The plates were developed for 1 h at 25°C. by the addition of 50 μl of 10 mM phenolphthalein monophosphate in0.2 M 2-amino-2-methyl-1-propanol, 0.5 M Tris, pH 10.2 with 0.1% sodiumazide. The reaction was terminated by the addition of 50 μl of 30 mMTris, pH 10.2, with 15 mM EDTA and the absorbance at 560 nm wasdetermined. The background value for this assay was generallyA560<0.060. Clones with absorbances 4-fold or greater above backgroundwere selected for further characterization.

Antibodies that displayed reactivity with H3396 cells were also analyzedfor binding to fixed HF235 normal human fibroblasts. HF235 cells werefixed 15 min at 25° C. in 2% paraformaldehyde in PBS after removing theculture medium. The cells were then washed four times with PBS prior touse. The background value for the ELISA with fixed fibroblasts wasgenerally A560<0.080. Minimal reactivity with the fibroblasts wasdefined as binding less than 2-fold above background.

In order to determine the isotype of the antibodies produced by thehybridomas, microtiter plates were coated with 0.5 μg/ml either goatanti-human IgM or goat anti-human IgG. Antibody binding was detectedwith goat anti-human Ig alkaline phosphate conjugate. Development of theassay was terminated when supernatant yielded a positive signal with oneof the capture antibodies. Likewise, the light chain class wasdetermined by capturing the antibody with the appropriate heavy chainreagent and detecting with goat anti-human γ or κ chain-specificalkaline phosphatase conjugate.

Immunoglobulin quantitation was performed similarly, except thatsupernatants containing unknown quantities of Igs were serially diluteduntil reactivity was undetectable. Ig concentrations were calculatedfrom values of dilutions that fell within the linear range of thestandard curve, defined by standard samples of purified polyclonal humanIgM, used in the range of 0.01 to 2.0 μg/ml.

Immunoglobulins were precipitated from LH11238 hybridoma supernatant bycentrifugation at 800×g for 10 min and clarification by filtrationthrough a 0.45 μm cellulose acetate filter. The supernatant was placedin dialysis tubing (M_(r) cut-off 12-14 kDa) and concentrated two- tofour-fold by coating the tubing with Aquacide (Calbiochem) and placingit at 4° C. for 6-8 h. Excess Aquacide was removed, the dialysis bagrinsed, and the sample was dialyzed 12-24 h versus multiple changes ofice-cold distilled water. The precipitated antibody was collected bycentrifugation at 10,000×g for 30 min. The supernatant was removed andthe pellets were resuspended in a minimal volume of warm 10-foldconcentrated PBS. Following solubilization of most of the pellet, thebuffer concentration was adjusted to PBS and the insoluble material wasremoved by centrifugation at 10,000×g for 30 min. Antibodies H1140,H2420 and H935 could similarly be precipitated in low ionic strengthbuffer.

The LH13 antibody was not precipitated in low ionic strength buffer.Therefore, in order to obtain concentrated antibody the volume of theclarified supernatant was reduced with an Amicon apparatus utilizing aYM-30 membrane. The antibody was concentrated greater than 50-fold bythis approach.

Five percent of the hybridomas screened (372/7216) bound tumor surfaceantigens greater than four-fold above background. Of these 372 clones,55 (15%) produced antibodies that did not bind fibroblastssignificantly. Although numerous antibody-secreting hybridomas wereisolated from control MLRs that had not been incubated with tumor cellsor membrane fractions, none of these antibodies displayed preferentialtumor reactivity. Approximately 20% (11/55) of hybridomas producingantibodies that displayed tumor specificity were stable through multipleexpansions in culture. Five of these clones secreted greater than 20μg/ml antibody. The properties of these clones are summarized in Table2. All five antibodies were of the IgM isotype and displayed a range ofimmunoreactivities as determined by assaying on fixed monolayers ofH3396 cells. At least one antibody was generated from each of theculture conditions and immortalization procedures employed. The twoantibodies displaying the greatest immunoreactivity, LH13 and LH11238,were characterized further (below).

The properties of the tumor-specific human monoclonal antibodiesproduced by hybridoma cell lines H1140, H2420, H935 LH11238 and LH13 aresummarized in Table 3. TABLE 3 Characteristics of tumor-specific humanmonoclonal antibodies. Isotype/ Secretion Light chain Level Immuno-Hybridoma class (μg/ml)^(a) Reactivity^(b) Precipitation^(c) H1140 IgM/λ40 1.0 + H2420 IgM/λ 63 1.3 + H935 IgM/λ 36 1.4 + LH11238 IgM/κ 29 5.7 +LH13 IgM/λ 26 341 −^(a)Secretion level is typical value obtained from supernatant of aterminal culture^(b)Immunoreactivity (ΔA560/[(μg Ig) (min)]) determined on confluentparaformaldehyde-fixed H3396 monolayers and normalized toimmunoreactivity of H1140^(c)Precipitation denotes ability (+) or inability (−) to preciptitateantibody in low ionic strength buffer

In summary, this example shows that an unstimulated human immunerepetoire (splenocytes from non-tumor-bearing patients) can be used togenerate human monoclonal antibodies reactive with novel tumor antigens.This was achieved through the stimulation of MLR cultures with eitherwhole tumor cells or membrane fractions derived from tumor cells. Thespecificity of the antigen stimulation was demonstrated by thegeneration of tumor-specific antibodies as well as by the absence oftumor-specific antibodies from cultures not treated with tumor cells ormembrane fractions.

EXAMPLE II Immunoreactivities of LH13 and LH11238 Antibodies

This Example shows the immunoreactivities of LH13 and LH11238 antibodieswith a panel of human tumor cells.

In order to use LH13 and LH11238 antibodies for immunodiagnostic andimmunotherapeutic purposes, the range of tumor types expressing thecorresponding antigens needs to be identified. Tumor cell lines arerepresentative of the corresponding tumor type, and normal fibroblastsare representative of non-neoplastic tissues. The tumor cell lines werederived from melanomas, lung carcinomas, ovarian carcinomas and breastcarcinomas. The human monoclonal antibodies of the invention were testedfor immunoreactivity with both human cancer cell lines and normalfibroblasts.

H3396, H3464, H3477 and H3922 are cultured cell lines established frommetastases of human breast adenocarcinoma, which were explanted andmaintained in culutre. H2981 and H2987 are cultured cell linesestablished from human lung carcinomas, which were explanted andmaintained in culture. H3639 and H3723 are culture cell linesestablished from human ovarian carcinomas, which were explanted andmaintained in culture. Each cell line was derived from a differentexplant.

Immunoreactivities were determined by the ELISA procedure describedabove, by incubating a broad range of antibody concentrations with fixedmonolayers of tumor cells. Antibody binding was measured with saturatingquantities of detect antibody. Antibody binding was measured as theΔA560/[(μg Ig)(min)] in the linear range of the assay. Theimmunoreactivities of tumor-specific human monoclonal antibodies againsta panel of tumor and normal cells are presented in Table 4. TABLE 4Immunoreactivity of human monoclonal antibodies LH13 and LH11228 againsttumor and normal cells. Immunoreactivity (ΔA560/ [(μg Ig) (min)]) CellLine Description LH13 LH11238 HF285 normal fibroblast 0 0 H2669 melanoma0 0 H3774 melanoma 0 0 H3396 breast carcinoma 1.136 0.019 H3464 breastcarcinoma 0.507 0.022 H3477 breast carcinoma 0.046 0.008 H3922 breastcarcinoma 0^(a) 0 H2981 lung carcinoma 0.584 0 H2987 lung carcinoma 0 0H3639 ovarian carcinoma 0^(a) 0.011 H3723 ovarian carcinoma 2.602 0.026^(a)Exhibited substantial immunoreactivity upon permeabilization ofcells with 0.1% digitonin

As shown in Table 4, LH13 displayed a broad cross-reactivity on thepanel of tumor cells with high (H3723 and H3396), intermediate (H2981and H3464), and low (H3477) immunoreactivities. The LH13 antigen wasabsent or present at undetectable levels on intact normal fibroblasts(HF285), melanomas (H2669 and H3774), and with several of the intactcarcinomas tested (H3922, H2987, and H3639). The lack of reactivity withH3922 cells was particularly surprising because the LH13 antibody wasisolated from lymphocytes which had been stimulated with H3922 cells. Toexamine this more closely each of the cell lines was permeabilized with0.1% digitonin and their reactivity with LH13 was re-examined. Two ofthe cell lines which were completely negative when intact cells wereassayed, H3922 and H3639, bound the LH13 antibody under conditions inwhich the antibody had access to intracellular compartments.Furthermore, H2981 cells, which had an intermediate LH13immunoreactivity when assayed under non-permeabilizing conditions werehighly reactive under permeabilizing conditions (immunoreactivitygreater than 7.0). The data obtained for immunoreactivities in thepresence and absence of 0.1% digitonin could not be compared directly,however, due to differential cell loss from the cell culture dishesduring the incubations.

LH11238 displayed a similar reactivity profile, though the magnitude ofits-immunoreactivity was always much less than that of the LH13antibody. Reactivity was observed with intact H3396, H3464, H3477,H3639, and H3723. Other cells tested were negative, including thefibroblast and melanoma cell lines. Based on the immunoreactivitydeterminations made for each of the cell lines with LH13 and LH11238 itappears unlikely that these antibodies recognize the same epitope. Forinstance, LH13 displayed moderate immunoreactivity with the H2981 lungcarcinoma line while no binding of LH11238 to this cell line wasdetected. Likewise, LH11238 bound intact H3639 ovarian carcinoma cells,even though binding of LH13 could not be detected under identicalincubation conditions.

EXAMPLE III Binding Activity of LH13 and LH11238 Human MonoclonalAntibodies

This Example shows the binding activity of human monoclonal antibodiesLH13 and LH11238 with normal and tumor cells.

In order to determine the immunoreactivity of human monoclonalantibodies with live tumor cells and normal cells, flow cytometry (FACSanalysis) was used. H3464 breast tumor cells were chosen because theyexpressed both LH13 and LH11238 antigens by ELISA analysis and werereadily isolated as a single cell suspension. Normal peripheral bloodlymphocytes were also examined as representative of normal tissues. Facsanalysis additionally permits examination of heterogeneity of a cellpopulation with respect to antigen expression.

H3464 cells were removed from culture dishes with trypsin or Versene(EDTA), washed with PBS, resuspended at 2×10⁶ cells/ml in tumor media,and allowed to recover 2 h at 37° C. Antibodies were incubated at 5.0μg/ml with 1×10⁶ tumor cells in 50 μl total volume for 30 min on ice.The cells were washed once with 1.0 ml of ice-cold PBS, incubated with 2μg/ml FITC-labeled goat anti-human IgM diluted in 1% BSA-PBS for 30 minon ice, and washed once more with 1.0 ml of ice-cold PBS. Antibodybinding to cells was analyzed with a Becton Dickinson FACSort.

Consistent with the ELISA results, H3464 cells incubated with LH11238and FITC-labeled antihuman IgM displayed a shifted staining patternrelative to an irrelevant control human IgM (FIG. 1A). Greater than 90%of the cells tested bound LH11238 antibody, though the broad range offluorescent intensities observed was consistent with heterogeneousexpression levels of the antigen on these tumor cells. Similar FACSstaining profiles were also observed with H935, H2420, and H1140.

Surprisingly, LH13 antibody, which was 23-fold more reactive thanLH11238 on H3464 cells as determined by ELISA (Table 4) displayed littleshift (FIG. 1B). To determine if the LH13 antigen was particularlysensitive to the proteolytic conditions used to isolate the tumor cellsfor analysis (trypsin), cells were allowed longer recovery periodsfollowing isolation or non-proteolytic cell isolation methods wereutilized, such as Versene (EDTA) release. Neither of these approachesresulted in significantly greater staining of the tumor cells.

Facs analysis of normal human cells, such as peripheral bloodlymphocytes, was negative for each of the antibodies tested, consistentwith the ELISA results obtained with normal fibroblast cells.

EXAMPLE IV Characterization of LH11238 Antigen

This Example describes the subcellular characterization of the LH11238antigen.

Both the ELISA screening and the FACS analysis of live cells describedabove demonstrated the expression of LH11238 antigen on the plasmamembrane of tumor cells. However, these approaches did not examine thedistribution of the antigen on intracellular structures ofcarcinoma-derived cells. To examine the intracellular localization ofLH11238 antigen, immunofluorescence analysis was used.

Monolayers of H3464 cells were seeded on 12-mm round coverslips (No. 1thickness, 0.06-0.13 mm thick) one day prior to use and were fixed with2% paraformaldehyde in PBS for 15 min at 25° C. The cells were rinsedtwice with PBS and incubated with 50 μl/ml antibody diluted either in 1%BSA-PBS (non-permeabilized) or in 1% BSA-PBS containing 0.1% digitonin(permeabilized) for 2 h at 4° C. The cells were rinsed twice with PBSand were then incubated in the dark with FITC-labeled goat anti-humanIgM diluted 1:500 in the same buffer as the primary antibody. The cellswere rinsed 4 times with PBS and the coverslips were mounted inFluoromount-G (Southern Biotechnology). Cells were visualized with anOlympus microscope equipped with epifluorescent optics and an OlympusSplan 40× (NA 0.70) objective lens.

Non-permeabilized, paraformaldehye-fixed H3464 cells stained with theLH11238 antibody displayed surface staining consistent with localizationto the plasma membrane. Consistent with the FACS analysis, greater than70% of fixed H3464 cells bound LH11238. When H3464 cells werepermeabilized to allow access to intracellular structures, punctate,peri-nuclear staining was observed. LH11238 binding to H3464 cells wasspecific as control incubations with irrelevant, isotype andconcentration-matched human antibodies did not stain intact or 0.1%digitonin permeabilized cells. The punctate, peri-nuclear stainingobserved on permeabilized cells suggested localization of the antigen inthe lysosomes. To verify this, cells were stained with antibodies toCD63, a known lysosomal glycoprotein. Incubation of H3464 cells withantibody to CD63 stained intracellular structures similar to thoselabeled with LH11238, consistent with a lysosomal localization ofintracellular LH11238.

Based on these results it was concluded that the LH11238 antigen ispresent both on the plasma membrane and in the lysosomes of H3464 cells.

EXAMPLE V Internalization of LH11238 Antigen

This Example shows that LH11238 antigen is internalized from the plasmamembrane to lysosomal compartments.

Immunofluorescence experiments described above indicated that LH11238antigen was present on both the cell surface and in lysosomes of tumorcells. The dual localization could result from LH11238 antigen beingco-expressed in lysosomes and the cell surface, or from beinginternalized to endosomal/lysosomal compartments. In order to determinewhether LH11238 antigen was internalized, the procedure forimmunofluorescent localization was modified.

Monolayers of live H3464 cells were chilled on ice for 30-60 min tocompletely inhibit endocytosis. The cells were then washed, incubatedwith LH11238 antibody, washed again, and incubated with FITC-labeledgoat anti-human IgM antibody. The cells were maintained at 4° C.throughout all of the preceeding steps to ensure complete inhibition ofendocytosis. The cells were then shifted to 37° C. with pre-warmed cellculture media. At various intervals the cells were shifted back to 4° C.and fixed with 2% paraformaldehyde. Initially, diffuse surface stainingwas observed, identical to that observed with non-permeabilized fixedcells. Following 10 min at 37° C., clustering of the LH11238 antibody atmultiple sites on the cell surface was observed. With longer incubationtimes, LH11238 first localized to a few sites on the cell surface, andthen began to internalize. These staining patterns were not observed ifcells were incubated with irrelevant primary antibodies, incubated withsecondary antibody only, or if the cells were maintained at 4° C. forthe duration of the experiment. These results are consistent withLH11238 binding and being internalized by H3464 cells.

In summary, the LH11238 antibody was first characterized as a surfaceantigen based on ELISA analysis of intact cells, FACS analysis of livecells, and immunolocalization using non-permeabilized cells. However,further immunolocalization studies with permeabilized cells demonstratedthat a portion of this antigen was also localized to a punctate,peri-nuclear compartment. Similar staining was observed with cellsincubated with anti-CD63 antibody, a lysosomal protein. Consistent withthe dual localization of the LH 11238 antigen on plasma membranes and inthe lysosomes, it was demonstrated that antibody bound to the surface oflive cells was internalized. The dual localization of tumor antigens tothe plasma membrane and lysosomes has been observed previously. Forinstance, the BR96 antibody binds the lysosomal-associated membraneprotein lamp-1 on the surface of intact tumor cells, even though lamp-1is normally an integral membrane protein predominantly located in thelysosomal compartment. In addition, the secretion of elevated levels ofsoluble lysosomal proteins, such as cathespins B, D, and L from tumorcells has also been documented. A portion of the secreted cathespin D isbound by the mannose 6-phosphate receptor and internalized. At present,it is unclear whether LH11238 is soluble or an integral membraneprotein. However, the antigen is found in the soluble fraction of aTX-114 phase separation of cell extracts, consistent with LH11238 beingmembrane-associated as opposed to an integral membrane protein.

EXAMPLE VI Characterization of LH13 Antigen

This example shows the localization of the LH13 antigen.

The LH13 antibody exhibited significant immunoreactivity when assayed byELISA on fixed monolayers of carcinoma-derived cell lines, as describedabove. However, FACS analysis suggested that LH13 bound only poorly tocell surfaces. Further ELISA analysis indicated that LH13 antigen waspredominantly present in a compartment that was only accessible toantibody when the cells were treated with 0.1% digitonin. To furtherexamine the expression of LH13 antigen in either subcellularcompartments or in the secreted medium, immunofluorescence analysis anddirect ELISA analysis were used.

To examine the subcellular localization of LH13 antigen,immunofluorescence was performed on paraformaldehyde-fixed H3464 cellsas described above. Incubation of LH13 antibody with unpermeabilizedH3464 cells (as described above using LH11238 antibody) resulted in weakstaining of the cell surface, consistent with the slight shift observedby FACS analysis. Incubation of LH13 antibody with permeabilized H3464cells by immunofluorescence resulted in no detectable staining.Possibly, little LH13 is associated with intracellular structures inH3464 cells or intracellular forms of LH13 were not recognized by theantibody in this cell line.

These results could also indicate that LH13 antigen was predominantlysecreted. To observe secreted LH13 antigen, a constant amount of LH13antibody (0.01 μg/ml) was diluted into increasing amounts of culturemedia which had been removed from confluent monolayers of H3396 cells(conditioned media). Conditioned media was then assayed for binding tofixed H3396 monolayers. A modest reduction (approximately 15%) in thebinding of the LH13 antibody to the monolayer was observed withincreasing concentrations of conditioned media (FIG. 2A), consistentwith LH13 antigen being present in the media. No reduction in thebinding of the LH13 antibody to the monolayer was observed withincreasing concentrations of conditioned media from H3922 cells.

To observe LH13 antigen in conditioned media of H3396 cells, a directELISA format was used. Conditioned media was incubated in a 96-well cellculture dish for 10-12 h at 4° C., removed, and the washed wells weresubsequently assayed for LH13 antibody binding. The antigen present inconditioned media obtained from H3396 cells bound to the culture dishand was readily detected (FIG. 2B). The specificity of antibody bindingwas demonstrated by incubating LH13 antibody with cell culture wellsthat had been pre-treated with: (1) conditioned media from a tumor lineexpressing undetectable quantities of LH13 (H3922), (2) fresh media(media), or (3) nothing (blank). None of these pre-incubation conditionsresulted in detectable LH13 binding to the well (FIG. 2B).

In summary, characterization of the LH13 antigen demonstrated that themajority of this antigen is found in conditioned media. Based on thisobservation, it was concluded that LH13 is secreted from tumor cells.

EXAMPLE VI Purification and Further Characterization of LH13 Antigen

This example describes the purification and further characterization ofLH13 antigen.

As described above, the LH13 antigen is secreted from breast tumor cellsand displays a surprisingly high affinity for cell culture dishes. Theseobservations suggested that LH13 antigen could be readily purified fromconditioned H3396 media using its binding to culture dishes as a meansof monitoring its purification. Once the LH13 antigen is purified, itssusceptibility to various reagents could readily be determined tofurther characterize its properties.

As a first step in enriching the LH13 antigen, the H3396 cell line wasadapted to grow in serum-free media supplemented with minimal protein[Iscove's media supplemented with TCH (Celox, defined serumreplacement), 2 mM L-glutamine, non-essential amino acids, and 1 mMsodium pyruvate]. Cells grown under these conditions secreted comparablelevels of the antigen as grown in the presence of serum. Conditionedmedia was collected, pooled, and 250 ml was diluted to 1 liter withwater to reduce the ionic strength. The diluted media was applied at 3ml/min to a 11 cm×1.5 cm column of Q SEPHAROSE FAST FLOW agarose resinwhich had been equilibrated with 0.25-fold concentrated (0.25×) PBS. Themajority of the antigen bound to the column as demonstrated by the lackof reactivity of the flow-through fraction. After washing the columnwith 200 ml of 0.25× PBS, protein was eluted at a flow rate of 6 ml/minwith a 90 min linear gradient from 100% 0.25× PBS to 70% 0.5 M NaCl in0.25× PBS.

Column fractions were assayed for protein content with the BCA proteinassay using BSA as the standard. Column fractions were assayed for LH13antigen as follows. Each fraction was diluted 10-fold into water and 50μl/well was transferred to a sterile 96-well cell culture dish for 12 hat 4° C. The plate was washed twice with PBS and incubated with 10 μg/mlLH13 antibody diluted in 1% BSA-PBS for 2 h at 37° C. The plate was thenwashed four times with PBS, incubated with goat anti-human lg alkalinephosphatase conjugate diluted 1000-fold into 1% BSA-PBS for 1 h at 25°C., and was developed as described above.

Most of the protein eluted between 150 mM and 250 mM NaCl (FIG. 3,closed circles), while the majority of the LH13 antigen eluted above 250mM NaCl (FIG. 3, open circles). The specific activity (ELISAsignal/[protein]) of the peak antigen fraction was 200-fold greater thanthe starting material. As a control, LH13 antigen was diluted with arange of NaCl concentrations (50 mM to 400 mM) to determine if the ionicstrength affected binding to the cell culture dishes. The range of NaClconcentrations employed for binding and eluting LH13 from the column didnot affect antigen binding to the cell culture dish. Therefore, thedirect ELISA accurately reflects the distribution of antigen in thevarious column fractions.

Column fractions were further resolved by electrophoresis on 4-20%SDS-polyacrylamide gradient gels. Coomassie blue staining revealed asingle major protein in the most reactive fractions, but Western blotsof the various column fractions failed to identify this as theLH13-reactive antigen.

To further characterize the LH13 antigen, its susceptibility to avariety of treatments was examined using the direct ELISA method. LH13antigen was coated on cell culture dishes, as described above, andincubated under a variety conditions. LH13 antigen was incubated with 5mg/ml trypsin at 37° C. for 30 min. Alternatively, LH13 antigen wasdenatured by treatment with 10% SDS+2% β-mercaptoethanol at 37° C. for30 min. prior to treatment with 2 U/ml sialidase (Oxford GlycoSystems),400 U/ml endoglycosidase-F/peptide-N-glycosidase F (Endo F, PNGaseF)(Oxford GlycoSystems), 60 mU/ml endo-α-N-acetylgalactosaminidase(O-glycanase)(Oxford GlycoSystems), or 2 U/ml sialidase plus 60 mU/mlendo-α-N-acetylgalactosaminidase (Sialidase, O-glycanase) at 37° C. for24 h. The culture dishes were washed three times with PBS and assessedfor LH13 antibody binding as described above. The effect of eachtreatment was compared to control samples which were treated underidentical conditions with buffer alone (FIG. 4).

Treatment of LH13 antigen with 10% SDS±2% β-mercaptoethanol, sialidase,or endo-α-N-acetylgalactosaminidase (O-glycanase) had little effect onsubsequent binding of antibody. The resistance of the antigen totreatment with sialidase and O-glycanase was not affected by denaturingthe antigen prior to treatment nor by simultaneous treatment with bothglycosidases. Binding of antibody in the direct ELISA was reduced bytreatment with trypsin (79%, FIG. 4) or withendoglycosidase-F/peptide-N-glycosidase F (36%, not shown). The bindingof antibody to LH13 denatured with 10% SDS+2% β-mercaptoethanol prior totreatment with endoglycosidase-F/peptide-N-glycosidase F was furtherdiminished, reducing binding by 75% as compared to untreated samples(FIG. 4).

Due to the broad range of specificities ofendoglycosidase-F/peptide-N-glycosidase F it was not possible to reachspecific conclusions regarding the structure of the carbohydrate.However, binding of LH13 antibody was completely unaffected by treatmentwith sialidase, which removes N- or O-acyl, non-reducing terminal sialicacids. Treatment with endo-α-N-acetylgalactosaminidase, which removesGalβ1-3GalNAc associated with serine or threonine, or the combination ofsialidase and endo-α-N-acetylgalactosaminidase also did not affectantibody binding. Trypsin sensitivity of antibody binding to antigensuggested that the epitope is associated with a protein component.

In summary, LH13 antigen was purified more than 200-fold from H3396serum-free conditioned medium, using anion exchange chromatography.Preliminary studies indicated that purified LH13 antigen is excludedfrom a Superdex 75 gel filtration column, consistent with the antigenbeing larger than 70 kDa. Treatment of LH13 antigen bound to cellculture plates with a variety of reagents provided evidence that bothcarbohydrate and protein components are present, and are involved ineither binding of the epitope to LH13 antibody or binding LH13 antigento tissue culture plates. At present, it is not possible to distinguishbetween these two possibilities.

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. A human monoclonal antibody or functional fragment thereof,comprising at least one Complementarity Determining Region (CDR) havingsubstantially the amino acid sequence of a CDR of SEQ ID NO:2 or SEQ IDNO:4.